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Benedique C.

Valdez

BS-Chemistry III

February 10, 2015

UNIVERSITY OF SAN CARLOS


DEPARTMENT OF CHEMISTRY

Name: Benedique C. Valdez


10, 2015
Course: BS-Chemistry III
_____________________

Date:

February

Approved
Exercise No. 3

Enzymes
Abstract
Enzymes are naturally occurring biomolecules in the bodies of both plants
and animals. They are essential because they are said to be the biological
catalysts in bodily reactions. Enzymes are found all-through-out the bodies of
living things. Examples of these enzymes are amylases and catalases.
Amylase is found in the saliva and is responsible for the breakdown of
complex starch molecules to simpler glucose units. Catalase on the other
hand is present in both animal and plant cells and is responsible for the
fixation of toxic hydrogen peroxide in both. The difference lies in the optimal
temperature of the enzymes depending on the source. Animal catalase from
liver have a higher optimal temperature than that of the potato filtrate
catalase which can be attributed to the normal temperature of the bodies of
the sources. Not only temperature but pH can dictate the activity of an
enzyme. Certain enzymes act optimally at neutral pH like catalase while
others like pepsin will be optimally active at low pH. Another factor that
dictate the activity of enzymes in biological systems is the presence of
inhibitors which either compete with the substrate of interest or destroy the
form the active site of the enzyme by binding in another part.
Introduction
Enzymes are abundant in all living organisms as they are biological catalysts
for bodily reactions. Due to its biological nature, these enzymes are
constrained by certain factors. With the aid of this experiment, the factors
that dictates the enzymatic activities can be represented with a series of
results in support to the literature data available. Also, this experiment could
lead to the discovery of side reactions and thus limiting factors to the usage

Benedique C. Valdez

BS-Chemistry III

February 10, 2015

of certain chemicals to demonstrate the factors affecting enzymatic


activities.
Experimental Details
Part I: Activity of Enzymes
Amylase
Prior to collection of saliva, the mouth of the source was washed. Two
milliliters of saliva was collected from the source in a 50 mL beaker. Ten
milliliters of 1% starch solution was gathered in a test tube. Two test tubes
were prepared labelled as Test tube 1 and Test tube two containing 1 mL of
the starch solution with 2 drops of iodine solution and 1 mL of starch solution
with 1 mL of Fehlings solution A and 1 mL of Fehlings solution B. Test tube 1
was set aside. Test tube 2 was then heated in a water bath for 5 minutes.
Observation was gathered.
The remaining (8 mL of starch solution) was mixed with 2 mL of
collected saliva in another test tube. It was shaken well to ensure
homogeneity. It was then maintained in a water bath at 37C for thirty
minutes. After, the solution was then halved and tested with the same
solutions (iodine and Fehlings solutions). Observation was gathered.
Catalase
A small potato was pared and grated into fine pulp. The pulp was the
mixed with 100 mL of ice water, left standing for 15 minutes, then filtered
through a cheese cloth. The filtrate gathered was then separated into two
parts in two test tubes. One of the half was boiled in a water bath. A few
drops of 3% H2O2 was then added into each test tubes.
Part II: Factors Affecting the Activity of Enzymes
A. Effect of Temperature
Separate test tubes containing 1 mL of pork liver extracts were
prepared. Each test tubes were immersed in a water bath following
the order: Test tube 1 (ice bath at 0C-5C), Test tube 2 (37C-40C),
Test tube 3 (boiling water bath). After 15 minutes of exposure to the
temperatures, 1 mL of hydrogen peroxide was added to each test
tube without removing the vials from the baths. Avoid shaking the
mixtures. The height of the foam was measured in centimeters after

Benedique C. Valdez

BS-Chemistry III

February 10, 2015

5 minutes of standing. A graph was constructed with the y-axis as


foam height and the x-axis as the temperature.
B. Effect of pH
Separate test tubes containing 1 mL of pork liver extracts were
prepared. Each test tubes were treated as follows: Test tube 1 (1 mL
of 1M HCl and 1mL of hydrogen peroxide), Test tube 2 (1 mL of
hydrogen peroxide), Test tube 3 (1 mL of NaOH and 1mL of
hydrogen peroxide). The foam height was then measured in
centimeters after 15 minutes of standing. Avoid shaking the
mixtures. A graph of pH versus foam height (x versus y) was then
made.
C. Effect of Inhibitor
Separate test tubes containing 1 mL of pork liver extracts were
prepared. Each test tubes were treated as follows: Test tube 1 (1 mL
of 95% ethanol), Test tube 2 (1 mL of 0.1 M mercury(II) nitrate
solution), Test tube 3 (1 mL of distilled water). All three test tubes
were immersed in a water bath maintained at 37C for 5 minutes.
Each of the test tubes were treated with 1 mL of hydrogen peroxide.
The height of the foams generated was compared per test tubes.
Results and Discussion
Amylase is an enzyme found in saliva specifically called salivary amylase.
This enzyme catalyzes the breakdown of starch into smaller sugar units like
glucose (-D-glucose). Due to this reason, the test tubes untreated with
amylase showed a positive result with iodine in KI solution (indigo solution)
and negative with Fehlings solutions (no brick red precipitate; no formation
of CuO2). This means that starch is present in both solutions for starch will
form a blue-violet starch-iodo complex. However, upon treating the solutions
with salivary amylase fresh from the source and maintaining it at 37C for 30
minutes, the exact opposite results occurs. The solutions treated with
amylase showed positive result for Fehlings solution upon heating (brick red
precipitate formed; CuO2 present) which tells us that there is a reducing
sugar present in solution. The other solution treated with iodine solution did
not form any blue-violet color, signifying that there was no starch present in
solution. Therefore, the enzyme salivary amylase did act on the starch
present in solution which is the substrate, and formed glucose as a product.
Another diverse type of enzyme present in both plants and animals is the
catalase. It is present in humans primarily in the liver for fixing toxins. In

Benedique C. Valdez

BS-Chemistry III

February 10, 2015

plants, potatoes for example, this enzyme is used in fixing toxic metabolism
side-products, specifically hydrogen peroxide. This enzyme also act the same
in humans livers to that in plants. The reaction of the substrate, hydrogen
peroxide, with the enzyme catalase goes:
3H2O2

catalase

2H2O

O2

However, due the fact that enzymes themselves are proteins, they are very
sensitive to temperature changes. Some of these enzymes have their
optimum temperatures at low temperatures such as the potato catalase.
Heating the enzyme solution would cause denaturation of the enzymes and
thus destroying their secondary, tertiary, and quaternary structures. Without
these structures, the enzymatic action of proteins are deactivated or
removed and thus minimal or absolutely zero enzymatic activity on hydrogen
peroxide can be observed. This is shown in the reaction of heated potato
filtrate with hydrogen peroxide which yielded minimal bubbles (O2 (g)) as
product.
As mentioned earlier, enzymes are proteins themselves. As biological
catalysts, they have their specific and optimum conditions for optimal
activity towards their specific substrates in the biological systems. Pork liver
extract like the humans liver, contains catalase which fixes the toxic
metabolism hydrogen peroxide side-product. Temperatures are to be specific
for each enzyme according to their optimal temperatures for optimal activity.
Pork liver will be on its optimal activity when the normal body temperature of
a pig is attained upon reaction with hydrogen peroxide, theoretically. This is
supported by the data gathered from the experiment. The test tube
maintained at 37C had the highest foam height among the three treatments
for it is close to the normal body temperature of a pig at 38.739.8. The
treatment maintained in an ice bath still had enzymatic activity lower than
that of the optimally treated one. The test tube held in a boiling water bath
had zero enzymatic activity (no bubbles present) because the catalase in the
solution was already denatured due to intense heating.
pH is another constraint for enzymatic activity. Experimentally, the result
were as follows (in decreasing order of enzymatic activity): base treated,
neutral solution, acid treated. According to literature, however, the optimal
pH of catalase in pork liver is around pH 7 or neutral solution. This can be
rationalize by focusing on the side reaction of hydrogen peroxide and sodium
hydroxide which yields a gaseous product and thus entails a higher foam

Benedique C. Valdez

BS-Chemistry III

February 10, 2015

height than the neutral solution. The reaction of hydrogen peroxide and
sodium hydroxide goes:
2NaOH

H2O2

Na2O2 (g)

2H2O

Still, the neutral solution did have a relatively high enzymatic activity
compared to the zero activity presented by the acidic solution. Theoretically
the only solution that should have had foam present could have been the
neutral solution.
Unlike catalase, pepsin helps in the digestion of proteins in the stomach at
pH 2. Therefore, the optimal pH for pepsin must be pH 2. Upon entering the
intestinal part of the human body system, which has a pH of 8, the
enzymatic activity of pepsin is hindered and thus digestion of proteins by
pepsin is lowered or ultimately stopped.
Another factor that limits the activity of enzymes are the presence of
inhibitors. Inhibitors are substances that tend to lower the enzymatic activity
towards the specific substrate of interest by blocking the active site of the
enzyme disabling the substrate of interest to bind on the site. Ethanol and as
well as heavy metals (mercury(II) nitrate solution) are inhibitors. They tend
to bind with the enzyme themselves and denatures the enzyme disabling its
enzymatic activity towards the substrate which is hydrogen peroxide. Heavy
metals denature proteins in a higher degree than ethanol as what was
observed in the experiment. This is rooted to the nature of denaturation of
both substances. Heavy metals such as mercury denatures the protein by
blocking the active site of the enzyme and or by binding itself in another site
of the protein and consequently altering the form of the active site of the
enzyme. The combined effects of these two results to a greater degree of
denaturation caused by heavy metals. Ethanol, on the other hand, only
disrupts the hydrogen bonding of the proteins which results to relatively
lower denaturation effect to the protein and thus lower inhibiting effect than
heavy metals.
Conclusion
Enzymes are biological catalysts and therefore are biomolecules that are
very sensitive to the pH, temperature, and the presence of inhibitors. These
enzymes are important in each and every living creatures as they hasten the
reactions and bodily processes. Without these biological catalysts, life could
be impossible. For example, without catalase, hydrogen peroxide would not
be metabolized to water and oxygen in cells leading to poisoning in cells and

Benedique C. Valdez

BS-Chemistry III

February 10, 2015

ultimately death of the creature. These enzymes are under the category of
proteins in the list of general biomolecules. Due to this nature, enzymes like
any other proteins, are sensitive to pH, temperatures, and inhibitors. Most of
these enzymes, are programmed by their source ,which is the thing from
which they came from, to do their specific tasks at a specific set of
conditions. Catalase of example is present in both animals and plants. The
optimal temperatures of the catalases from both differ. Plants for example,
have a generally lower optimal temperature for their catalases compared to
animal catalases. This can be attributed to the differences of the body
temperature of the sources. Plants have a relatively colder body temperature
than animals so their enzymes are set to act optimally on a lower
temperature than that of animals. pH also is another condition which can
alter enzymatic activity. Pork liver extract contains catalase which fixes
hydrogen peroxide optimally at pH 7. Due to the side reaction of sodium
hydroxide and hydrogen peroxide, however, it showed the highest enzymatic
activity which is a false positive as the activity was not fully attributed to the
enzymatic activity of catalase to hydrogen peroxide but as a combination
with the side reaction. Another factor limiting the activity of enzymes is the
presence of inhibitors. These substances can inhibit by blocking the path of
the substrate to the enzyme which deforms the active site such as heavy
metals which greatly inhibits the enzymatic activity relative to the action of
ethanol which only disrupts hydrogen bonding. All of these behaviours can
be summarized and pointed out into one single cause, which is the nature of
enzymes: enzymes are proteins.
References
Campbell, Mary and Farrell, Shawn. "Chapter 6: The Behaviour of Proteins:
Enzymes" Biochemistry. 7th edition, International Edition ed. Pacific Grove, CA:
Brooks/Cole Pub., 2012. N. pag. Print.

Normal Rectal Temperatures,


http://www.merckmanuals.com/vet/appendixes/reference_guides/normal_rect
al_temperature_ranges.html